Exam June 2017, questions and answers PDF

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Examination Candidate Number: _____________ Desk Number: _____________

University of York Department of Biology B. Sc Stage 3 Degree Examinations 2016-17 Epigenetics in Development and Disease Time allowed: 2 hours Total marks available for this paper: 100 This paper has two parts: Section A: Short Answer / Problem / Experimental Design questions (50 marks) ● Answer all questions in the spaces provided on the examination paper Section B: Essay question (marked out of 100, weighted 50 marks) ● Answer either question A or question B ● Write your answer on the separate paper provided and attach it to the back of the question paper using the treasury tag provided ● The marks available for each question are indicated on the paper ● A calculator will be provided

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SECTION A: Short Answer / Problem / Experimental Design questions Answer all questions in the spaces provided Mark total for this section: 50

1. Human dermal fibroblast (HDF) cells were used to create either induced pluripotent stem (iPS) cells or nuclear transfer embryonic stem (NT-ES) cells following somatic cell nuclear transfer. Global DNA methylation analysis was performed on the iPS and NT-ES cells and compared with ES cells derived from in vitro fertilization (IVF ES) to identify differentially methylated regions. The differentially methylated regions were then compared with the original HDF cells. The data is presented below. Stem Cell Type

Number of methylated regions that are different to IVF ES cells

Number of differentially methylated regions that are shared with HDF cells

iPS cells

6478

780

NT-ES cells

110

87

This question was based on lecture 4 material (LO: Understand why somatic cell nuclear transfer and induced pluripotent stem cell production must involve epigenetic re-programming) a) What does this data suggest regarding the efficiency of the two different reprogramming techniques in terms of erasing the DNA methylation profile of the original HDF cells. (2 marks) It suggests that SCNT is more efficient at erasing the DNA methylation profile of the original HDF cell compared with the iPS technique. This is evidenced by the relatively fewer differences between NT-ES cells and IVF ES cells and between NT-ES cells and HDF cells compared with iPS cells. Most students got this correct. b) Suggest an explanation for the observed difference in the efficiency of DNA methylation reprogramming by iPS and NT-ES production. (5 marks) SCNT transfers a somatic nucleus into an enucleated oocyte which then undergoes the normal developmental programme (1). This should page 2 of 11

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involve large scale erasure and resetting of DNA methylation patterns (1) so NT-ES cells are likely to resemble normal ES cells (1). iPS production could be argued as being a more artificial method with forced expression of transcription factors (1) driving pluripotency by gene expression changes - thus retaining the methylation pattern of the HDF cells is more likely (1). Variable answers with the most common mistake being failure to understand SCNT. It relates to content of lecture 4 and required understanding of how SCNT and iPSC are performed and linking this to the natural erasure of DNA methylation that occurs post-fertilization. LO3: Discuss the importance of epigenetic mechanisms at key points in development LO7: Demonstrate understanding of epigenetic mechanisms and key techniques through interpreting data from primary research papers 2. In order to investigate whether the enzyme TET1 is required for the erasure of genomic imprints during primordial germ cell development, Tet1 knock out male mice were created. These were mated with wild-type female mice and produced low birth-weight offspring with small placentas compared with offspring of a control mating. This question was testing understanding of imprinting and the ability to apply what was known about Tet1 activity in the zygote to germ cell development. L5 LO: Understand that genomic imprinting in mammals involves the establishment of parent-specific DNA methylation marks in the gametes that escape re-programming in the early embryo. a) Outline a hypothesis that would explain this observation. (4 marks) Tet1 is involved in removing DNA methylation at imprinted genes during paternal primordial germ cell development (1). Loss of Tet1 results in smaller offspring and placenta due to reduced expression of paternally expressed growth promoting genes (1) as a consequence of failure to remove methylation marks (1). This relates to content in L5 and understanding imprinting. Mostly good answers with some linking to specific imprinting examples.

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The DNA methylation status of the Peg10-Sgce imprinting control region (ICR) was then assessed in two individual offspring of the paternal Tet1 knock-out (PaternalKO  ) compared with two control offspring (Ctrl). The figure below is data obtained by standard bisulfite sequencing of two regions within the ICR. It shows methylated and unmethylated cytosines as filled and unfilled circles, respectively. The percentage of DNA methylation is indicated.

b) Briefly describe how bisulfite sequencing allows the DNA methylation status of cytosines to be determined at single-base resolution. (3 marks) The bisulfite treatment converts cytosines to uracil (1) but leaves methyl-cytosine as cytosine (1). Because the treatment is followed by sequencing (cloned PCR products or by next generation methods) information on the methylation status of individual cytosines can be determined (1). Mostly very good answers. The most common mistake was to suggest that methylated cytosines will convert to uracil. c) Provide an explanation for the patterns of methylation observed in the control offspring compared with the paternal Tet1 knock-out (PaternalKO  ) offspring. (4 marks)

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In the control the pattern shows approximately half the DNA strands are fully methylated and half are largely unmethylated (1). The methylated strands are most likely maternal in origin whereas the unmethylated strands are paternal (1). In the PaternalKO  there is almost complete methylation likely due to failure to erase the methylation at this region during paternal primordial germ line formation (1). Most students got some marks for this question though many didn’t consider the 50:50 pattern of methylation shown in the control. As stated in the introduction to the question, the experiment is aimed at investigating whether the enzyme TET1 is required for the erasure of genomic imprints during primordial germ cell development. However many students answered this question thinking about TET1 activity in the zygote. The cross was between a TET1 defective male and a wild-type female so TET1 would be present in the zygote and the imprints escape erasure anyway at that point. d) Predict the outcome if the bisulfite analysis was performed on offspring of a maternal Tet1 knock-out mated with a wild-type male. Provide an explanation for your prediction. (3 marks) Prediction is that the offspring will have a similar 50:50 pattern as the control cross (1).This region is normally maternally methylated and so the loss of Tet1 activity should not matter (1). The paternal allele will be unmethylated as Tet1 activity will remove the methylation mark during germ cell development (1). Most students got the answer correct but with the wrong explanation. LO3: Discuss the importance of epigenetic mechanisms at key points in development LO7: Demonstrate understanding of epigenetic mechanisms and key techniques through interpreting data from primary research papers 3. The figure below is ChIPseq data showing levels of H3K4me3, H3K27me3, H3K9me3 or the H3K9 methyltransferase SETDB1, across a region of human chromosome 7 that includes the Cebpa locus. Cebpa encodes a transcription factor expressed in adipocytes (fat cells). Data is shown from Embryonic Stem Cells (ESCs) and preadipocytes (an intermediate stage between ESCs and adipocytes). The input track controls for non-specific immunoprecipitation. The numbers below Chr7 indicate ChIPseq levels. The numbers to the right of Chr7 indicate the locus position on the chromosome and are not important for the

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question.

a) Discuss whether the data provides evidence of bivalent chromatin at the Cebpa locus in ESCs and/or preadipocytes. (4 marks) Yes there is evidence in both however the patterns are different and it could be argued that only ESCs show true bivalency (2). In ESCs there is overlapping regions of active (H3K4me3) and inactive (H3K27me3) marks (1). The situation in preadipocytes also shows regions of active and inactive marks (in this case H3K9me3) but these do not overlap (1). This question tested whether students understood what is bivalent chromatin (L3 LO: Understand that stem cells have characteristic chrmatin organisation.). Mostly good answers here though sometmes confusion regarding whether a mark was active or not. b) Use the data to predict whether Cepba is likely to be expressed in preadipocytes and state how you would test this experimentally. (3 marks) The presence of H3K9me3 (repressive mark) across the coding region suggests that it wouldn’t be expressed despite the H3K4me3 in the upstream region (2). This could be tested via rtPCR (other appropriate techniques credited) (1) Mostly very good answers. c) What do you predict will be the profile of H3K4me3, H3K27me3,

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H3K9me3 and SETDB1 across the Cepba locus if the experiment was repeated on adipocytes? Justify your answer. (4 marks) Given that Cepba is expressed in adipocytes we would predict that the repressive marks (H3K9me3,H3K27me3) and also SETDB1 (responsible for H3K9me3) would be reduced/disappear (2). The H3K4me3 will be retained (1) but could spread out across the region as there is some evidence from the data that H3K9me3 is restricting it (compare profile in ESCs and preadipocytes) (1). A common mistake was to think that Cepba would be switched off (though in the question it says that it is expressed). Good answers in general though. d) It is proposed that SETDB1 is recruited to the Cepba locus via the methyl-cytosine binding protein MBD1. Describe how you could test this experimentally. (4 marks) In preadipocytes first use ChIP to assess whether MBD1 is present at the Cepba locus and whether this profile overlaps with that of SETDB1 (2). Then knock-down expression of MBD1 by siRNAs (1). Repeat ChIP to see whether this has affected SETBD1 levels at the Cepba locus (1). Mostly good answers. A common mistake was to only consider whether SETDB1 interacts with MBD1 and not to include whether this interaction was involved in recruiting SETDB1 to the locus. LO1: Describe how DNA methylation, histone modifications and non-coding RNAs influence gene expression LO3: Discuss the importance of epigenetic mechanisms at key points in development LO7: Demonstrate understanding of epigenetic mechanisms and key techniques through interpreting data from primary research papers 4. Bladder cancer cells were continuously treated with 10−4  M zebularine (a derivative of 5-azacytidine) for up to 40 days. Samples were harvested at the indicated times and protein lysates were resolved in a Western blot (A). Relative protein expression levels were quantified (normalised to PCNA levels) (B). mRNA levels were assessed using RT-PCR (C). DNA methylation levels of various hypermethylated tumour suppressor loci in these cells were also quantified (D).

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a) Describe how zebularine influences DNMT levels by referring to the data in figures A-C above. (4 marks) The effect of zebularine is at the protein level as mRNA levels are not affected (1). Zebularine specifically causes degradation of DNMT1 protein (1). Levels of DNMT3a protein are temporarily reduced, but recover within 40 days (1). DNMT3b3 are also reduced in the short term and remain at ~70% of initial levels at 40 days (1). Almost all students answered this question at least partially correctly. To receive full marks, figure B should be described quantitatively. And that Zebularine works at protein level (and from the lecture degradation/trapping) should have been highlighted.

b) Using the data explain why zebularine can be used as an effective cancer therapy drug. (2 marks) Zebularine results (based on Fig part D) in the reduced methylation of specific loci (tumor suppressors) which will result in the increased expression of these genes, ultimately inhibiting proliferation. LO5: Discuss why epigenetic mechanisms can influence disease, particularly cancer LO7: Demonstrate understanding of epigenetic mechanisms and key techniques through interpreting data from primary research papers Some students described how Zebularine works generally rather than referring to the experiments (Fig D). 5. Describe the changes to histone modifications that occur in cancer cells. (4 marks) In 'normal' cells, genomic regions that include the promoters of tumour suppressor genes are enriched in histone- modification marks associated with active transcription (1), such as acetylation of H3 and H4 lysine residues H4K16 and trimethylation of H3K4. Also DNA repeats and other heterochromatic regions are characterized by trimethylation of H3K27 and dimethylation of H3K9, and trimethylation of H4K20 which function as repressive marks (1). In cancer cells, this is disrupted by the loss of the 'active' (1) histone-marks on tumour-suppressor gene

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promoters, and by the loss of repressive (1) marks such as the trimethylation of H4K20 or trimethylation of H3K27 at subtelomeric DNA and other DNA repeats. This leads to a more 'relaxed' chromatin conformation in these regions. Specific histone modifications are not required to achieve full marks. Most common mistake here was a confusion between DNA methylation and histone modification. 6. Describe the epigenetic changes that occur in ageing twins. (4 marks) Experiments using monozygotic twins (1) showed an epigenetic drift (1) in aged twins in DNA methylation (both hypo- and hypermethylation)(1), and histone H4 and H3 acetylation (1) as compared to younger twins. LO5: Discuss why epigenetic mechanisms can influence disease, particularly cancer Many students answered this question correctly. Some of the mistakes: Needed to describe experiments that involved twins [monozygotic twin study mentioned in the lectures is the most obvious]. Needed to point out specific epigenetic marks. Some answered this question in terms of general epigenetic changes observed in ageing.

SECTION B: Essay question Answer one question on the separate paper provided Remember to write your candidate number at the top of the page and indicate whether you have answered question A or B Mark total for this section: 50

EITHER A. Discuss the mechanisms by which non-coding RNAs direct epigenetic change. Answer is expected to include examples of both long and short ncRNAs

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and to highlight similarities and differences in mechanisms. Examples of long ncRNAs that could be considered include HOTAIR, HOTTIP, XIST, AIR, H19 in mammals, COOLAIR and COLDAIR in plants. Answer should discuss the role of these RNAs as scaffolds to recruit histone modifying enzymes and chromatin remodellers. For example HOTAIR recruits both PRC2 components and histone demethylases to switch off gene expression. It acts in trans - though how it is recruited to its target is not well understood. HOTTIP acts in cis and aids chromatin looping, recruiting trithorax components to activate gene expression. Xist acts in X-inactivation, being expressed from and coating the inactive X. The step-wide activities should be described - removing active marks, exluding PolII and laying down repressive marks via PRC2 and DNMTs. How Xist is thought to link to and spread across the Xi can be discussed. Other long ncRNAs mentioned in the module include Tsix that is involved in controlling Xist expression and PolIV and PolV transcripts that are required for RNA-directed DNA methylation in plants. This pathway involves the generation of small non-coding RNAs that act as sequence-specificity determinants, guiding AGO and de novo DNA methyltransferases to target loci via and interaction with PolV scaffold transcripts. Extra reading could provide other examples, such as piRNAs as well as more mechanistic detail to the examples that were covered. Overall some very good answers with excellent detail and examples of further reading. A few essays were very limited in scope (i.e only described a couple of ncRNAs) or failed to consider what the RNAs are doing. Good accuracy and most answers stuck to the question closely. LO1: Describe how DNA methylation, histone modifications and non-coding RNAs influence gene expression. LO2: Compare and contrast epigenetic mechanisms between plants and animals LO3: Discuss the importance of epigenetic mechanisms at key points in development LO4: Describe how epigenetic mechanisms are involved in genome defense OR B. Discuss whether the biggest influence on the DNA methylation status of an organism is likely to be developmental or environmental. A good answer should express an opinion regarding what the biggest influence is on the epigenome. Many examples covered in the module can be used here to provide supporting evidence in favour of a particular

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argument. There is stronger evidence in animals in support of developmental influence than environmental. In plants there is less change in DNA methylation over the course of development but good evidence of an environmental (stress) impact. It is important that the supporting evidence and examples match the hypothesis appropriately. There is no need to cover all the possible examples. A good answer should include a discussion of the epigenome profiles of organisms - e.g. that the default state of mammals is to methylate CpG dinucleotides and that CpG islands are protected from this. This is greatly influenced by development stage especially in terms of the two waves of demethylation and remethylation that occur in early development. DNA methylation profiles differ between cell types with CpG islands of some genes becoming methylated as a consequence of cell fate decisions. There are several examples of environmental influence on the epigenome from the lecture (including agouti mouse, other examples of effect of diet, and insect and plant models) that can also be used to evaluate the influence of the environment.

LO3: Understand the importance of epigenetic mechanisms at key points in development LO6: Critically evaluate examples of how the environment can influence the epigenome Almost all students who answered this question expressed an opinion (either in favour of environmental effects or developmental effects). In some cases the evidence given did not support the argument very well, or mechanistic details were not clear or missing. In some cases the answer included irrelevant or not entirely relevant detail. A few answers included histone modification or ncRNA instead of or in addition to DNA methylation.

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